Defining the complex involvement of the transcription factor c-Myb in the regulation of haemopoietic stem cells and progenitors using conditional gene deletion

The c-Myb transcription factor, originally identified as an oncogenic variant in avian leukaemia viruses that transform immature haematopoietic cells, is expressed in haemopoietic stem cells (HSCs), committed blood cell progenitors, and at key stages during the differentiation of specific blood lineages. Constitutive deletion of the c-myb gene leads to embryonic lethality when definitive blood cells fail to develop in the foetal liver. To investigate the function of c-Myb in haemopoiesis we have generated knockdown and conditional alleles of c-myb. Mice homozygous for the knockdown allele are profoundly anaemic and immunodeficient, but interestingly exhibit thrombocytosis. The latter phenotype is also seen in several c-Myb mutants isolated in ENU-mutant screens. Deletion of c-myb in mice homozygous for the conditional floxed allele employing the interferon-inducible MxCre transgene led to a profound loss of bone marrow cells. Marrow hypoplasia was preceded by a rapid loss of erythroid progenitors and HSCs followed closely by an increase in megakaryocyte lineage cells.

Detailed analysis of erythroid cells from c-myb knockdown animals revealed that low levels of c-Myb lead to an accumulation of CD71+Ter119- cells expressing low levels of c-Kit but with no significant effect on proliferation. By culturing specific sorted populations of erythroid progenitors we could demonstrate that low levels of c-Myb retard their further differentiation. Upon deleting c-myb in cultured erythroid progenitors carrying the floxed allele and an interferon-inducible Cre-encoding transgene we observed a rapid decline in c-kit protein and RNA and the induction of globin gene expression, although obvious terminal differentiation was not seen. In contrast to cells expressing low levels of c-Myb, complete loss resulted in cell cycle arrest. Global gene expression changes in response to induced c-myb inactivation assessed by microarray screening revealed several novel c-Myb target genes.

A similar investigation of the consequences of reducing or eliminating c-myb expression in HSCs has revealed a novel role for c-Myb in their commitment to differentiation. Hence, in vitro culture of c-myb knockdown and conditionally c-myb deleted HSCs showed that most cells committed to megakaryocyte differentiation and terminally matured within two days. The aberrant commitment of HSCs when c-Myb activity was reduced was accompanied by changes in the expression of transcription factors and growth factor receptors previously implicated in HSC maintenance, indicating that c-Myb plays a pivotal role in the control of the stem cell. c-kit, long suspected as a possible c-Myb-regulated gene, has emerged as a potentially key target for the actions of c-Myb in HSCs as well as erythroid progenitors.